Measurements Of Protein Surface Verify Electrostatics Model

Date:

December 15, 1998

Source:

University Of Illinois At Urbana-Champaign

Summary:

Using a surface-force apparatus, researchers at the University of Illinois have measured the electrostatic properties of a protein surface at the molecular level. Their results provide the first direct comparison between localized measurements and theoretical predictions.

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CHAMPAIGN, Ill. - Using a surface-force apparatus, researchers at the University of Illinois have measured the electrostatic properties of a protein surface at the molecular level. Their results provide the first direct comparison between localized measurements and theoretical predictions.

"We obtained direct measurements of the pH-dependent electrostatic charge density of a single binding face of the protein streptavidin," said Deborah Leckband, a U. of I. professor of chemical engineering. "Our measurements show excellent agreement with values predicted from theory, thus verifying the accuracy of our measurement technique."

Identifying the electrostatic properties of a protein surface is useful for interpreting biochemical data and for obtaining a better mechanistic understanding of the forces that govern a protein's behavior. Many researchers believe, for example, that complementary charge distributions can generate steering torques that act like tiny tractor beams that pull proteins into the right orientation for binding.

To probe the local surface charges, Leckband, biophysics professor Shankar Subramaniam and graduate research assistant Sanjeevi Sivasankar first prepared homogeneously oriented monolayers of streptavidin by anchoring the protein to a supported lipid bilayer.

Using a surface-force apparatus, they then measured directly the electrostatic surface potential of the protein monolayer at a variety of pH levels. They were thus able to isolate the point of zero charge for the binding face of the protein. Importantly, the measured value for the exposed protein surface differed from the point of zero charge for the net protein.

"The difference in the pH-dependence between the probed surface and the soluble protein clearly demonstrates that these force measurements indeed reflect the local charge density of the oriented protein, rather than its net charge," Leckband said.

The experimentally measured surface-charge densities were then compared with theoretical predictions of the electrostatic potential distribution around the protein surface.

"The calculated values agreed very closely with those obtained by the surface-force measurements," Leckband said. "This tells us not only that we can measure the local properties of protein surfaces at the molecular level, but also that current models are reasonably accurate."

While the study focused on the pH-dependence of electrostatic surface-charge densities, "this direct approach to probing the electrostatic features of proteins is applicable to investigations of any perturbation that alters the electrostatic composition of the surfaces of immobilized macromolecules," Leckband said.

The researchers announced their findings in the October issue of the Proceedings of the National Academy of Sciences.

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